Abstract All neurodegenerative diseases, including Alzheimer?s disease (AD), are associated with the accumulation of misfolded protein aggregates. This proposal is rooted in the discovery of the brain?s glymphatic system, a brain- wide perivascular fluid transport system analogous to the lymphatic system in peripheral tissues, which clears protein waste products. Since a hallmark feature of aging and AD is a sharp reduction in glymphatic amyloid-? and tau clearance, we propose that degeneration of the glymphatic-lymphatic drainage plays an essential role in the pathogenesis of Alzheimer?s. Aim 1 will be to define, for the first time, the major efflux pathways of the glymphatic-lymphatic system based on uDISCO clearing, which renders whole rodent bodies transparent. The advantage of uDISCO clearing is that the efflux paths can be visualized without removing the brain from the skull ? a procedure that necessarily severs the glymphatic-lymphatic connections. Fluid-dynamic modeling based on real-time imaging of fluorescent tracers will provide a global map of the glymphatic-lymphatic flow patterns and their driving forces. Aim 2 will be to determine why glymphatic-lymphatic fluid transport is sharply reduced in aging and AD. The experiments will focus on identifying the bottlenecks that hamper clearance of amyloid-? and tau in aging and AD. Also, the distribution of immune cells (microglia, T-cells, and more) in CNS and meningeal lymphatic vessels will be mapped as a function of aging and AD after uDISCO clearing. Aim 3 will be to determine whether improving the quality of sleep delays aging and AD-induced degeneration of the glymphatic-lymphatic system. Non- pharmacological improvement of sleep architecture will test whether the long-term benefit of improved sleep quality is a result of delaying age-induced and AD-induced degeneration of glymphatic-lymphatic amyloid-? and tau clearance. We will test whether insomnia treatments, including non-benzodiazepine receptor agonists, GABAB receptor agonists, or sedating antidepressants, improve ? or perhaps worsen ? AD progression. To our knowledge, this proposal constitutes the first systematic analysis of the efflux pathways of the glymphatic- lymphatic system. The proposed research leverages very recent developments in tissue clearing and light-sheet microscopy techniques. The studies will address key gaps in our understanding of the glymphatic-lymphatic system, and will use state-of the art approaches to investigate what we believe is one of the most fundamental unexplored avenues ? and opportunities ? in CNS pathophysiology. The team of investigators has broad and complementary relevant expertise, including analysis of the glymphatic system, invention of the whole body uDISCO clearing, and fluid-dynamic analysis and modeling. Our goal is to create a complete map of the glymphatic-lymphatic connections in young, middle aged, and old wildtype and APP/PS1 mice and to identify age-related and AD-related bottlenecks in the clearance of amyloid-? and tau.